Anti ifn γ

C57BL/6 mice were inoculated with 5 × 10⁵ B16 cells, and at days 6 and 10, the mice were treated with saline or 10 µg mRNA encoding Fluc, tBid, or MLKL. Two days after the second treatment, spleens were isolated and passed through 70 µm nylon strainers (BD Biosciences, San Diego, CA, USA) to obtain single-cell suspensions. Red blood cells were lysed using ACK red blood cell lysis buffer (BioWhittaker, Wakersville, MD, USA) and 2.5 × 10⁵ cells were cultured for 24 h in wells of anti-IFN-γ (Diaclone, Besancon, France) pre-coated 96-well plates in the presence of 10 µg/ml peptide. The following synthetic, high-performance liquid chromatography-purified peptides were used for restimulation: OVA 257–264 (SIINFEKL), OVA 323–339 (SQAVHAAHAEINEAGR), CT26-M20 (PLLPFYPPDEALEIGLELNSSALPPTE), CT26-M26 (VILPQAPSGPSYATYLQPAQAQMLTPP), CT26-M03 (DKPLRRNNSYTSYIMAICGMPLDSFRA), CT26-M37 (EVIQTSKYYMRDVIAIESAWLLELAPH), CT26-M27 (EHIHRAGGLFVADAIQVGFGRIGKHFW), B16-M30 mut (PSKPSFQEFVDWENVSPELNSTDQPFL), and B16-M30 WT (PSKPSFQEFVDWEKVSPELNSTDQPFL).